1. Technical Field
The embodiments disclosed herein relate to automated systems for processing blots used in the field of molecular biology.
2. Introduction
Western blotting is one example of an immunostaining technique used extensively for over 30 years in biology laboratories, in order to detect one or more target proteins in a sample. A schematic of the Western blot procedure is shown in FIG. 1. First, the sample is loaded onto a gel and the proteins are electrophoretically separated in the gel, e.g., an SDS-PAGE gel, or a non-denaturing gel. (FIG. 1, step 1). The protein(s) within the gel are transferred, or blotted, onto filter paper or other membrane, either by capillary forces, or by electrophoresis, or blotted onto a piece of filter paper, e.g., polyvinylidene difluoride (PVDF) or nitrocellulose (FIG. 1, step 2). Protein binding to the filter is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. As such, the filter paper possesses non-specific protein binding properties in that it generally binds all proteins substantially equally.
After transfer, the filter paper is treated, or blocked, to prevent non-specific binding of proteins at subsequent steps. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein, such as bovine serum albumin or non-fat dry milk. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces “noise” in the final product of the Western blot, leading to clearer results, and eliminates false positives.
The detection of the target protein(s) is achieved either in a one-step or two-step process. In the two-step process, the blocked filter is treated with a primary antibody specific to the target protein, followed by treatment with a secondary antibody specific for the primary antibody, and which includes a detectable moiety. A dilute solution of primary antibody (generally between 0.5 and 5 μg/mL) is incubated with the membrane under gentle agitation. The antibody solution and the membrane are incubated together for anywhere from 30 minutes to overnight. It can also be incubated at different temperatures, with warmer temperatures being associated with more binding, both specific (to the target protein, the “signal”) and non-specific (“noise”).
The membrane is rinsed or washed to remove unbound primary antibody, and then incubated with a secondary antibody specific for the primary antibody and that contains a detectable moiety, which can be detected, as an indicator of the presence and/or amount of target proteins present in the original gel. The secondary antibody is incubated with the membrane for a period of time, with gentle agitation.
Alternatively, in the one-step process, the blocked membrane can be incubated with a primary antibody that contains a detectable moiety, thereby eliminating the necessity for a secondary antibody.
Treatment of the blot with the primary antibody, and, if required, secondary antibody and developing solution to visualize the detectable moiety, requires several steps of addition, incubation and washing, spread out over several hours. Automation of the Western blot development steps would advantageously reduce time and labor for processing samples. However, automation of Western blotting presents unique challenges, due to the nature of the reagents used. Specifically, proteins such as antibodies are susceptible to degradation and are therefore generally kept in a cold solution until they are ready for use. Further, antibodies are generally expensive, and, in many cases are in limited supply. Thus, it is desirable to avoid using a larger volume of solution, requiring larger volumes of antibody, in developing a Western blot. There is thus a need for a system that can reduce the need for manual manipulation of the Western blot, and that minimizes reagent waste.